There is a fundamental gap in our understanding of how focal adhesion kinase (FAK) is activated by activators at the cell membrane and how FAK regulates its translocation into the nucleus. FAK activation and nuclear translocation are two key contributors to pancreatic tumor growth and invasion. Therefore, this gap in knowledge is an important problem because it severely hampers design of targeted therapeutic intervention. The objective of this R03 project is to use x-ray crystallography to reveal how FAK regulates its nuclear localization through an intramolecular interaction and how FAK binds to activators that activate FAK by promoting FAK autophosphorylation. The atomic details revealed by these crystal structures will be essential for achieving my long-term goal of understanding and therapeutically controlling the role of FAK in tumorigenesis. My central hypothesis is that both autophosphorylation and nuclear translocation of FAK are governed by a nuclear localization signal (NLS) located on FAK's noncatalytic band4.1-ezrin-radixin-moesin homology (FERM) domain. This hypothesis is formulated on the basis of my preliminary data showing that FAK's focal adhesion targeting (FAT) domain binds directly to this NLS, and published research showing that two activators that trigger FAK autophosphorylation also bind to this NLS. The rationale for the proposed research is that the atomic details of the FERM:FAT and FERM:activator interactions will allow us to understand how autophosphorylation and nuclear localization of FAK are regulated. The objective of this R03 proposal will be achieved through two specific aims: (1) Determine the crystal structure of the intramolecular FERM:FAT domain complex; and (2) Determine the crystal structure of the complexes formed between the FAK FERM domain and the activators c-Met and PIP2. For Aim 1, protocols established for my preliminary studies will be used to produce recombinant FERM and FAT domains. Available robotics systems will be used for crystallogenesis of the FERM:FAT complex. Structures will be determined using molecular replacement based on isolated FAT and FERM crystal structures established previously by us and other groups. For Aim 2, the FERM domain will be co-crystallized with fragments from c-Met and PIP2, which were previously shown to bind to FERM directly. The proposed research is innovative because it will provide the first mechanistic insights into how a single regulatory element of FAK controls two key events. This contribution will be significant because it will enable the design of pleiotropic inhibitory compounds that simultaneously target autophosphorylation and nuclear import of FAK. Moreover, by blocking nuclear import of FAK, those inhibitors would simultaneously block FAK's action on several proapoptotic factors (such as p53 and Mdm2). Thus, this project will lay the groundwork for a full-scale R01 proposal to develop pleiotropic PPI inhibitors against a most promising target in pancreatic cancer. PUBLIC HEALTH RELEVANCE: The proposed R03 project is relevant to public health because it will contribute mechanistic insights into how FAK promotes tumor survival and metastasis. The first two protein-protein interaction (PPI) inhibitors against FAK, in combination with chemotherapy, have recently been shown to be extremely promising drugs against pancreatic cancer. The proposed research is relevant to the PA Pilot studies in Pancreatic Cancer because its results will allow developing improved second-generation PPI inhibitors against FAK-mediated pancreatic tumorigenesis.